Identification and Characterisation of a Novel Tissue Barrier in Tendon Blood Vessels C
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European Cells and Materials Vol. 31 2016 (pages 296-311) DOI: 10.22203/eCM.v031a19 ISSN 1473-2262 THE BLOOD-TENDON BARRIER: IDENTIFICATION AND CHARACTERISATION OF A NOVEL TISSUE BARRIER IN TENDON BLOOD VESSELS C. Lehner1,4,*, R. Gehwolf1, J.C. Ek2, S. Korntner1, H. Bauer3, H-C. Bauer1,4, A. Traweger1 and H. Tempfer1 1Institute of Tendon and Bone Regeneration, Paracelsus Medical University, SCI-TReCS, Salzburg, Austria 2Institute for Neuroscience and Physiology, Department of Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden 3Department of Ecology and Evolution, University of Salzburg, Salzburg, Austria 4University Clinic for Trauma Surgery and Sports Injuries, Salzburg, Austria Abstract Introduction Tissue barriers function as “gate keepers” between different Tendons harbour a population of multipotent stem/ compartments (usually blood and tissue) and are formed progenitor cells, which are heterogeneous and could be by specialised membrane-associated proteins, localising identified in both peritendon and tendon proper. These cells to the apicolateral plasma membrane domain of epithelial are described to reside both in the vascular compartment and endothelial cells. By sealing the paracellular space, the and the tendon proper (Lui, 2013; Tempfer et al., 2009). free diffusion of solutes and molecules across epithelia and The niche of these cells is not yet fully understood. The endothelia is impeded. Thereby, tissue barriers contribute finely tuned molecular and cellular composition of this to the establishment and maintenance of a distinct internal niche appears to be crucial for stem cell differentiation and external environment, which is crucial during organ and maintenance (Bi et al., 2007; Katenkamp et al., 1976; development and allows maintenance of an organ- Tempfer et al., 2009; Zhang and Wang, 2013; Zhang et al., specific homeostatic milieu. So far, various epithelial and 2010b). So far, besides oxygen tension and mechanical endothelial tissue barriers have been described, including stimuli, few other niche-determining factors are known the blood-brain barrier, the blood-retina barrier, the including the composition and nano-/micro-structure of blood-testis barrier, the blood-placenta barrier, and the the extracellular matrix (ECM) (Bi et al., 2007; Rui et al., cerebrospinal fluid (CSF)-brain barrier, which are vital for 2011b; Tong et al., 2012; Wang et al., 2008; Zhang and physiological function and any disturbance of these barriers Wang, 2013). can result in severe organ damage or even death. Here, The internal milieu of the niche is severely changed we describe the identification of a novel barrier, located during tissue degeneration, trauma and injury, often resulting in the vascular bed of tendons, which we term the blood- in pathological alterations such as hyperproliferation, tendon barrier (BTB). By using immunohistochemistry, erroneous tendon cell differentiation, and calcification transmission electron microscopy, and tracer studies we (Oliva et al., 2012; Rui et al., 2011a). Therefore, it appears demonstrate the presence of a functional endothelial reasonable to suggest that rapid restoration and stabilisation barrier within tendons restricting the passage of large of the niche following an injury is paramount in initiating blood-borne molecules into the surrounding tendon and supporting the healing process. tissue. We further provide in vitro evidence that the BTB Healthy tendons are sparsely vascularised (Ahmed potentially contributes to the creation of a distinct internal et al., 1998; Hooper et al., 1984; Weidman et al., 1984; tissue environment impacting upon the proliferation and Zhang et al., 1990), particularly sheathed tendons – such differentiation of tendon-resident cells, effects which might as the flexor tendon of the hand – and depend on synovial be fundamental for the onset of tendon pathologies. fluid diffusion to provide nutrition (Hagberg et al., 1992; Jones et al., 2003; Lundborg et al., 1980; Manske and Keywords: Tendon vasculature, blood vessels, endothelial Lesker, 1985; Tempfer and Traweger, 2015). Between cells, barrier, tight junctions, tracer, vascular permeability, densely packed collagen fibre bundles continuous rows serum, tendinopathy. of tenocytes are found perfectly aligned with the collagen structure in direction of the strain (Kannus, 2000; Legerlotz et al., 2014), exhibiting an exceptionally low turnover rate, which may also account for the poor regenerative capacity of tendon tissue (Heinemeier et al., 2013). *Address for correspondence: In contrast, chronically inflamed tendons or tendons Christine Lehner, PhD which are recovering from injury are characterised by Institute of Tendon and Bone Regeneration hypercellularity and hypervascularity, due to increased Paracelsus Medical University – Spinal Cord Injury & proliferation of tenocytes and vascular cells (Andersson et Tissue Regeneration Center Salzburg al., 2011; Astrom and Rausing, 1995). In vitro, an increase A-5020 Salzburg, Austria in proliferation has been observed in isolated tendon Telephone Number: +43 662 2420-80867 cells when cultivated with serum or platelet rich plasma E-mail: [email protected] (Mazzocca et al., 2012; Wang et al., 2012). www.ecmjournal.org 296 C Lehner et al. The blood-tendon barrier So far, tendon vasculature has been investigated Proliferation assay in the context of tendon development, tendinopathies To examine the effect of serum on cell proliferation, human and vascularisation of tendon tissue grafts (Fenwick et TDCs were cultured in the presence of pooled human al., 2002). Little attention has been paid to the tendon serum (Sigma, Vienna, Austria, #H6914) at concentrations vasculature as a restrictive system impeding the passive of 10 %, 5 %, 1 % and without serum. Cells were counted transport of solutes and molecules across vessel walls and after 3 and 5 d. For an additional experiment, we cultured thereby controlling the homeostasis of the tendon-specific human tendon cells either with 10 % serum or without niche. Such influx and efflux control systems usually rely serum. Cells cultured without serum were supplemented on a sophisticated network of membrane-spanning proteins with serum on the eighth day. In parallel, serum was (e.g. occludin, claudins and junctional adhesion molecules) withdrawn from cells cultured with serum so far. Again, linked to a vast array of cytoplasmic proteins, which seal cell numbers were counted every second or third day until the paracellular space between neighbouring cells (Bauer cells reached confluence after two weeks. et al., 2014; Bauer et al., 2011). The aim of this study was to investigate the structural, Differentiation experiments functional and molecular characteristics of blood To assess the effect of serum on tendon cell differentiation vessels in human and mouse tendon tissue. By means potential, human TDCs were cultured with and without of transmission electron microscopy (TEM), RT-PCR, serum supplementation for one week. After this period, immunohistochemistry, and in vivo tracer studies we either Adipolife™, Chondrolife™ or Osteolife™ (LM- showed that the vascular lining of tendon vessels serves 0023, Lifeline Cell Technology, Frederick, MD, USA), as a size-selective diffusion restraint, reminiscent of the serum-free adipogenic, chondrogenic or osteogenic media, tissue barrier located in the cerebral microvasculature of respectively, were added to both cell types for two weeks. vertebrates (Abbott et al., 2010). Moreover, we cultivated Subsequently, 1) adipogenic differentiation was assessed tendon-derived cells with increasing serum concentrations, by Oil red O staining and quantitative RT-PCR (qRT- thus mimicking a barrier breakdown in vitro, and examined PCR) determining the expression level of peroxisome the effect on proliferation and differentiation. proliferator-activated receptor gamma (PPARγ), a regulator We propose that this blood-tendon barrier (BTB) is of adipocyte differentiation (Rosen and Spiegelman, 2001), imperative for the establishment of stable homeostatic 2) chondrogenic differentiation by Alcian blue, collagen conditions, thus potentially contributing to the regulation II and aggrecan staining, 3) osteogenic differentiation of tendon cell differentiation. by staining for alkaline phosphatase, qPCR on three osteogenesis markers (runt-related transcription factor 2, RUNX2; bone sialoprotein 1, BSP1; and parathyroid Materials and Methods hormone 1 receptor, PTH1R) and visualisation of calcium deposits by Alizarin S (Sigma, Vienna, Austria) staining. Sample collection In total, 14 human tendon samples (biceps tendons: ß-Galactosidase staining n = 9, 8 men, 1 woman; mean age 60.4 years, range 30 In order to exclude that lack of serum affects cell to 75, obtained from rotator cuff repairs; semitendinosus senescence, we performed a ß-galactosidase staining tendons: n = 3, 2 men, 1 woman; mean age 31.6 years, according to the protocol described by Dimri et al. (1995). range 18 to 55, obtained from anterior cruciate ligament To this end, cells were cultivated for 2 weeks with either reconstruction; and Palmaris longus tendons: n = 2, 1 Adipolife™ without serum, Adipolife™ with 10 % human man, 1 woman; mean age 56 years, range 37 to 75) were serum, MEM with human serum, Osteolife™ without obtained with patients’ informed consents as approved by serum or Osteolife™ with 10 % human serum. The cells the Salzburg ethics committee (approval #E-Nr. 1809). were fixed for 1 min with 3 %